The physical environment of the skeleton is known to play an important role in the establishment and maintenance of structurally competent bone. However, the mechanisms by which physical signals exert such biological effects are unclear. While biophysical signals elicit a variety of cellular responses in bone cells, the mechanism by which these responses are initiated is unknown. In this study we propose that annexin V (AnxV), a CA2+-dependent phospholipid binding protein, is a critical protein required for initiating mechanotransduction. AnxV has a number of attributes, which suggest that it is ideally suited for a role as a mechanoreceptor, possibly a mechanosensitive ion channel. These include the ability to function as a Ca2+ selective membrane ion channel, and the ability to interact with both extracellular matrix proteins such as collagen, and cytoskeletal elements such as actin. Hypothesis: our central hypothesis is that bone cells detect and transduce physical signals into biological responses via a mechanism requiring AnxV. Aims: Our goal is test this hypothesis by determining the role of AnxV in the response of bone cells to oscillating fluid flow, a physiologically relevant physical signal in bone. We will determine whether AnxV, in a role as a Ca2+ selective, mechanosensitive ion channel, is required for flow-induced Ca2+ influx, global increases in Ca2+i and ultimately gene expression in human osteoblast-like MG63 cells and murine osteocytic MLO-Y4 cells (Aims 1, 2 and 3). We will also determine whether expression and cellular location of AnxV are regulated by fluid flow (Aim 4). Significance: The long-term goal of these studies is to increase our understanding of how biophysical signals are selected and converted into an appropriate biological response by bone cells. Identification of critical "mechanotransducer" proteins may also provide novel targets for future therapeutic interventions in the fight against bone diseases such as osteoporosis.